Target specific antibody-superantigen conjugates and their preparation

ABSTRACT

A soluble antibody conjugate comprising an antibody linked to a structure which is recognizable by T-cells and has the ability to direct T-cells to lyse the target cell, which is recognized by the antibody. The conjugate is characterized by the structure being a superantigen. One important mode is a method for the lysis of target cells, wherein the target cells are contacted with a target cell lysis effective amount of the conjugate. The method of lysis is part of a potent treatment regime for cancer, autoimmunity, parasitic infestations and fungal, viral and bacterial infections The specification also describes modes such as the synthesis of the conjugate and pharmaceutical compositions and their manufacture.

This application claims priority as a continuation of U.S. applicationSer. No. 08/339,279, filed Nov. 8, 1994, abandoned, which is acontinuation of U.S. application Ser. No. 07/961,937, filed Jan. 14,1993, abandoned, which is based upon PCT application numberPCT/SE91/00496, international filing date Jul. 16, 1991, which is basedupon Swedish patent application numbers 9002484-5 and 9002479-5, bothfiled Jul. 20, 1990.

The present invention concerns antibody conjugates which are capable ofactivating cytotoxic T-cells (CTLs). The conjugates are useful fordestroying undesired cells that are associated with i.a. cancer forms,autoimmune processes, parasitic infestations and bacterial, viral andfungal infections.

BACKGROUND OF THE INVENTION.

Attempts have been made over the past years to use antibodies incombination with agents that directly exert a toxic effect on targetcells (cytotoxic agents, cytotoxins) in order to provide a selectiveaction on target cells and to prevent and minimize the non-specificeffect on other cells. The combinations suggested have ranged fromcovalently bonded complexes using linkage-providing molecules andnon-covalently bonded complexes to simple mixings (e.g. Ghose et al., J.Natl. Cancer Inst. 61(1979)657-676 and Carlsson et al., Biotechnology7(1989)567-73). Suggested cytotoxins-have been i.a. diphteria toxin,ricin, subunit A of ricin, gelonin, and Pseudomonas aeruginosa exotoxinA (Takeda Chemical Ind., EP-A-336,405 and Pastan et al., WO-A-88/00703,both of which have been cited in connection with the priorityapplication, SE-9002479).

With the advent of the hybridoma technology and the accompanyingavailability of monoclonal antibodies, it has been feasible to use theconcept of complexes between antibodies and cytotoxic agents to morespecifically direct the cytotoxic agents to the intended target cellpopulation.

In view of the recognized damaging effect of cytotoxic agents on othercells than target cells, one has suggested to replace the cytotoxicagents with immune stimulators that trigger T-lymphocytes and activateCTLs. Specific proposals have been concerned with antibodies conjugatedto

(i) antibodies that are directed against a T-cell receptor or compoundsthat are able to bind to a T-cell receptor (Mass. Inst. Techn.,EP-A1-180,171);

(ii) compounds, such as antigens, mitogens, other foreign proteins, andpeptides that activate cytotoxic T-cells (Neorex Corp., EP-A1-334,300);

(iii) MHC antigens, (Behringwerke AG, EP-A1-352,761);

(iv) antigens against which the individual to be treated has immunity,(Med. Res. Counc. WO-A-90/11779 (publ. 1990-10-18)); and

(v) an unnamed bacterial enterotoxin (Ochi and Wake, UCLA-Symposium:Cellular Immunity and the Immunotherapy of Cancers, Jan. 27-Feb. 3,1990, Abstract CE 515. page 109).

However, the immune stimulators suggested hitherto have been either toospecific or too general in their action. For instance classical antigensactivate only about 1 out of 10⁵ T-cells while mitogens are potentiallycapable of activating a majority of the T-cells.

It has been recognized that certain-agents mediate activation of amoderate ratio of T-cells; i.e. they activate T-cells at a relativelyhigh frequency, but far from 100% (Fleischer et al., J. Exp. Med.167(1988)1697-1707; and White et al., Cell 56(1989)27-35, both articlesbeing incorporated by reference). This type of agents are more effectiveactivators than classical antigens and they accordingly have been namedsuperantigens (for a review see Kappler and Marrack, Science248:705,(1990)). It has further been demonstrated (Dohlsten et al.,Immunol. 71(1990)96-100; and Hedlund et al., Cell. Immunol129(1990)426-34, both articles being incorporated by reference) that thesuperantigens known so far have the capacity to bind to MHC Class IImolecules on target cells and activate cytotoxic T-cells bearing theproper T-cell receptor V beta chain. The published data indicate thatthe MHC binding is a prerequisite for T-cell binding and activation tooccur. It can not be excluded that in the future superantigens will befound that act through a T-cell receptor V alpha chain or other surfacestructures only found on subpopulations of T-cells.

The immunomodulatory effect of the superantigen Staphylococcusenterotoxin A (SEA) has also been described by Platsoucas et al (CellImmunol. 97(1986)371-85).

Most of the presently known superantigens have earlier been recognizedas toxins and all of them have been of microbial origin. Staphylococcalenterotoxins for instance are enterotoxic and activate T-cells, and thetwo effects are discernible from each other (Fleischer et al., Cell.Immunol. 118(1989)92-101; Alber et al., J. Immunol 144(1990)4501-06; andInfect. Immun. 59(1991)2126-34).

It has previously been suggested to use superantigens in order to directCTL mediated lysis of cells carrying MHC Class II antigens (PharmaciaAB, WO-A-91/04053, publ. 1991-04-04). WO-A-91/04053 covers, but does notexplicitly mention, superantigens that are incorporated into covalentimmunoconjugates.

Cells lacking MHC Class II or expressing marginal amounts of MHC ClassII proteins do, however, not bind sufficient amounts of superantigens inorder to efficiently direct lysis of them by CTLS. Thus due to thegeneral abundance of cells carrying MHC Class II antigens and thenon-abundance of MHC Class II antigens on most tumour cells,superantigens should be of low value for the specific killing of suchunwanted cells.

However, we have found that a specific cell-killing effect mediated byCTLs can be achieved with superantigens, if they are covalently linkedto an antibody directed against an epitope that is specific for the cellto be killed. The activation of the immune system may induce targetcells lacking MHC Class II antigens to express them, which maypotentiate the desired lytic effect.

SUMMARY OF THE INVENTION

The present invention provides novel antibody conjugates

(i) comprising (1) an antibody directed against target cells, and (2) asuperantigen, i.e. a structure that is recognized (interact with or bindto) and activate T-cells, in particular CTLs;

(ii) methods for destroying target cells, in particular in connectionwith therapeutical treatment methods contemplated on mammals, and forspecific activation of T-cells, such as CTLs;

(iii) method of synthesis for the conjugates; and

(iv) pharmaceutical compositions containing the conjugates andpreparation methods for the compositions.

The methods for destroying target cells encompasses therapeutictreatment methods for cancer, autoimmunity, viral infections, bacterialinfections, fungal infections, parasitic infestations and other diseasesin which the objective is to kill certain cells with a high degree ofaccuracy. The conjugates of the invention may be used for themanufacture of pharmaceutical compositions intended to be used fordestroying target cells associated with the diseases just given. Theindividuals to be treated are normally animals, primarily human beings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEA-C215 mAb conjugate directing CTLs against MHC class IIcolon carcinoma cells.

FIG. 2 shows SEA-C215 mAb conjugate and SEA-C242 mAb conjugate inducedCTL targeting against colon carcinoma cells,

FIG. 3 shows lysis of SEA-C215 mAb conjugate coated colon carcinomacells mediated by SEA but not by SEB responding CTLs.

FIG. 4 shows cytotoxicity induced by SEA-C242 mAb conjugate andSEA-Anti-Thy-1.2 mAb conjugate against target cells.

DETAILED DESCRIPTION OF THE INVENTION Superantigen part of the conjugate

The novel antibody conjugates are characterized by the structure that isrecognized by the T-cells being a superantigen. The conjugates arenormally soluble at physiological pH and in vitro they are soluble inserum.

Preferred superantigens are selected from the group of staphylococcalenterotoxins (SEs), such as SEA, SEB, SEC, SED and SEE, toxoids, activefragments or peptides thereof and other substances having essentiallythe same mode of action for activating CTLs. The superantigens mayinclude other microbial products (bacterial as well as viral), such asproducts from staphylococcal strains, e.g. Toxic Shock syndrome toxins(TSST-1), from Streptococci, e.g. pyrogenic exotoxin A, and bacterialexoproteins and proteins produced by mycoplasma arthridis, which havesimilar capacity to interact with T-cells in the same way assuperantigens do. Superantigens may be obtained by culturing of theirnatural producers or of genetically engineered cells (recombinanttechniques) or potentially also by synthetic peptide synthesis. Asuperantigen to be used in the invention should, when applied to theexperimental model presented in this specification, exert effects inanalogy with what we present in the experimantal part of thisspecification.

The preferred superantigens have the potential ability to bind to:about1-40% of the isoforms of a polymorphic T-cell surface protein associatedwith the activation of CTLs, preferably the T-cell receptor V betachain.

The antibody part of the conjugate

The antibody is preferably a monoclonal antibody (mab) althoughpolyclonals may be used as long as they provide a sufficiently narrowspecificity. The expression antibody refers to antibodies in general andthus encompasses antibody active fragments, and other moleculesmimicrying the binding ability of an antibody, providing they have theappropriate specificity, avidity and affinity for the target cell inquestion. This includes genetically engineered (recombinantly produced)antibodies and antibody derivatives or other similarly bindingstructures. In the one embodiment the antibody is specific for anantigenic determinant on tumour cells, for instance a colon carcinomaassociated determinant (epitope, structure). It is also conceivable thatthe antibody may be specific for an antigenic determinant on cellsresponsible for autoimmunity, virus infected cells, bacteria, parasitesor fungi or other un-wanted cells. Depending on the efficiency of theconjugate, the antibody may be directed to an antigen that willinternalize the antibody after binding, although it is believed thatsuch antibody specificities are not preferred.

Monoclonal antibodies studied in connection with this invention are theC215 antibody directed against an antigen in the GA-733 family (see forinstance EP-A-376,746 and references cited therein and Larsson et al.,Int. J. Canc. 42(1988)877-82), the C242 antibody (Larsson et al., Int.J. Canc. 42(1988)877-82) and the Thy-1.2 antibody (Mab C, Opitz et al.,Immunobiol. 160(1982)438- ). It is conceivable that mabs havingspecificities for other target cell surfaces structures will be useful.The preparation of monoclonals directed against epitopes unique forselected target cells are wellknown in art. See for instance theabove-mentioned publications. Expressions, such as monoclonal antibodiesdirected against the C242 epitope or the C215 epitope, cover antibodiesreacting with cross-reacting epitopes.

Of the three monoclonals tested so far C215-conjugates most probablywill be of minor importance because they react with an epitope on atumour antigen that too frequently is expressed on normal cells.C242-conjugates appear to be better based on specificity data althoughour results indicate that they may require higher dosages. Mab Cdirected against Thy-1.2 will probably be of low value for targetinghuman cancer cells because the Thy-1.2 antigen is specific for anon-human mammalian tumour cell.

A hybridoma cell line producing the C242 monoclonal antibody has beendeposited on Jan. 26, 1990 under number ECACC 90012601 at EuropeanCollection of Animal Cell Culture, Porton Down, Salisbury,-Wilts, U.K.

The structure linking the superantigen to the antibody

In the preferred conjugate of the invention the superantigen iscovalently coupled to the antigen through a covalently linkage. (--B--).If it is important that the conjugate will not decompose whenadministered to the cells to be killed, for instance to an animal, thelinkage should be essentially metabolically stable for a sufficientperiod of time for the effect to be achieved. Furthermore, it is ofadvantage that the linkage as such does not give rise to immunologicalreactions itself. In general the linkage shall be inert in the sensethat the conjugate retains an efficient target cell binding specificity(anti-tumour, antiviral etc.) and an efficient capability of activatingcytotoxic T-cells.

In the scientific as well as in the patent literature several functionalgroups have been suggested in linkage structures of immunoconjugates.Accordingly the linkage --B-- in our novel conjugates may containstructures selected from the group consisting of

(i) amides and hydrazides (amides=--CONR₁ -- or --NR₁ CO--, where eachof the free valencies binds to a saturated carbon atom, and R₁ may behydrogen or an alkyl substituent such as lower alkyl (C₁₋₆) or thealpha-N-substituent of a naturally occurring alpha amino acid,preferably a hydrophilic amino acid, and hydrazides=--CONHNH-- or--NHNHOC-- where each of the free valencies binds to a saturated carbonatom);

(ii) thioether and disulfide (--S_(r) -- where each of the freevalencies binds directly to a saturated carbon atom, respectively, and Sis a sulphur atom, and r an integer 1 or 2);

(iii) straight, branched or cyclic hydrocarbon chains which aresaturated and which possibly may be substituted with one or more hydroxyor amino groups;

(iv) ether (--O--, where each of the free valencies binds directly to asaturated carbon-atom); and

(v) primary amine or disubstituted hydrazine (--NH-- or --NH--NH--,respectively, where each of the free valencies binds directly to asaturated carbon atom).

The length of the bridge should be within the ranges normallycontemplated within the technical field, i.e. shorter than 180 atoms,such as <100 atoms, but longer than 3-6, preferably longer than 16atoms.

The preferred linkage is hydrophilic and should not contain any aromaticring. Preferred hydrophilic structures that may form part of the linkage--B-- are: (i) polypeptide chains of the naturally occurring hydrophilicalpha amino acids (e.g. asparaginic acid and its amide, glutaminic acidand its amide, lysine, arginine, glycine, threonine, serine and possiblyalso histidine); (ii) oxaalkylene chains such as (--O(CH₂)_(n))_(n') -where n is an integer 2-5, preferably 2-3, and n' may be an integer1-20; and (iii) --S-- (thioethers), --O-- (ethers) and unsubstitutedamides (--CONH--) all of which being linked to short unsubstitutedhydrocarbon chains (C₁₋₄), preferably containing 1 or 2 carbon atoms.

Hydrophilic amino acids may be present in hydrophilic structures of thetype (F--(Pro)_(n))_(m) F, wherein F represents an amino acid sequence,preferably 4-8 residues, in which each amino acid is individuallyselected from serine, glycine and threonine, m is an integer 1-4 and nan integer 4-8 (Cetus Corp., WO-A-85/03508).

The linkage --B-- may be attached either at specific locations in theantibody or superantigen part of the conjugate or at random. Potentiallocations are an amino terminal, a carboxy terminal and a lysine residue(omega amino group). If the antibody or superantigen carries a thiolgroup or disulfide group (cystine or cysteine, respectively) thesegroups may also be used for covalently coupling, unless they are notessential for the activity of the active parts of the conjugate. Whenpresent, carbohydrate structures can be oxidized to aldehyde groups thatin turn may used for linking to the other moiety of the conjugate (cf.Cetus Corp., EP-A-240,200).

The conjugate of the invention should not contain any significantamounts of ester bonds and labile amide bonds, in particular not formedwith tyrosine and histidine residues, respectively. If such bonds havebeen formed during the synthesis they can be removed by use ofhydroxylamine (Endo et al., Cancer Res. 48(1988)3330-3335).

The number of superantigen moieties that may be present per antibodyactive moiety is normally 1-5, preferably 1 or 2.

In one of the preferred modes the conjugate substance shall besubstantially uniform with regard to superantigen per antibody, and/oremployed binding positions in the superantigen and antibody moieties,respectively, and/or linkage --B-- etc. In other words all theindividual conjugate molecules in conjugate substance should be the samein regard to these variables.

The substance should be essentially free from unconjugated antibodies orunconjugated superantigens.

The exact ratio superantigen to antibody, linkage structure etc. for theoptimal conjugate will depend on the selected monoclonal (includingclass, subclass, producing clone, specificity) and selectedsuperantigen. The experimental models given in this specification willenable the screening for optimal parameters also for other superantigensand other antibodies.

According to the embodiment of the invention studied most extensively upto the filing date, the linkage --B-- comprises the structure

    --SrRCONHCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m COY--                                                     (I)

The free valencies in formula I link to the active parts, respectively.This takes place either directly or through further divalent inertstructures that are comprised within the bridge --B--.

n is an integer >0, e.g. 1-20, preferably 2 or >2 and in many cases <10.m is 1 or 2.

S is a sulphur atom and binds directly to a saturated carbon atom ateach of its valencies (--S_(r) --=a thioether or a disulfide). r is aninteger 1 or 2.

Y is --NH--, --NHNH-- or --NHN═CH-- that at their left ends bind to theCO group shown in the right terminal in formula I and at their rightends to a saturated carbon atom or to a carbonyl group (only when Yequals --NHNH--).

R is preferably alkylene (having 1-4 carbon atoms, often 1 or 2 carbonatoms), that possibly is substituted with one or more (1-3, in thepreferred case <2) hydroxy (OH) groups.

Preparation of the antibody-superantigen conjugate

The antibody conjugates of the present invention can be obtained byenriching and purifying them from culture media of cells producing them,or from other media in which they have been synthesized.

The synthesis of our novel conjugates may be accomplished by techniquesknown in the art for conjugate synthesis, i.e. genetic engineering(recombinant techniques) or via the appropriate antibody andsuperantigen by classical coupling reactions at appropriate functionalgroups. The functional groups present in proteins and normally utilizedare:

(i) Carbohydrate structures. This structure may be oxidized to aldehydegroups that in turn are reacted with a compound containing the group H₂NNH-- to the formation of a --C═NH--NH-- group.

(ii) Thiol group (HS--). The thiol group may be reacted with a compoundcontaining a thiol-reactive group to the formation of a thioether groupor disulfide group. Free thiol groups of proteins are present in cystineresidues and may be introduced onto proteins by thiolation or splittingof disulfides in native cysteine residues.

(iii) Free amino groups (H₂ N--) in amino acid residues. An amino groupsmay be reacted with a compound containing an electrophilic group, suchas an activated carboxy group, to the formation of an amide group. Thefree amino group preferably is an amino terminal or the omega aminogroup of a lysine residue.

(iv) Free carboxy groups in amino acid residues. A carboxy group may betransformed to a reactive (activated) carboxy group and then reactedwith a compound containing an amino group to the formation of an amidegroup. However, precautions must then be taken to minimize amideformation with the amino groups that mostly are present together withcarboxy groups in the same protein. The free carboxy group preferably isa carboxy terminal or a carboxy group of a diacidic alpha amino acid.

The compounds carrying a H₂ NNH-- group, a thiol-reactive group, anactivated carboxy group, or an amino group may be bifunctional couplingreagents or an antibody or a superantigen. The groups are bound directlyto saturated carbon atoms except for the H₂ NNH-- group that as analternative also may be bound to a carbonyl carbon. The groups may havebeen introduced onto the the antibody or superantigen by commonderivatization.

Recombinant techniques provide efficient means for the manufacture ofconjugates in which the parts are specifically linked together from aterminal carboxy group in one moiety to a terminal amino group in theother moiety. A linkage structure consistent with the technique appliedmay be inserted. The reagents employed are selected so that they willprovide the linkage --B-- as defined above. Common bifunctional reagentshave the formula Z--B'--Z-- where Z and Z' are are functional groupsthat are mutually consistent with each other and allowing for covalentcoupling at a functional group present on a protein. See above. B' is aninert bridge that may contain the same structures as given for thelinkage --B-- above. Particularly Z and Z' may be identical or differentand selected among a thiol group, a thiol-reactive group, an activatedcarboxy, --CONHNH₂ etc. For a definition of these groups se below underthe heading Novel Reagents.

The method we have used for the conjugates employed in the experimentalpart comprises the steps of:

(i) reacting the antibody or the superantigen with an organic reagentcontaining a thiol-reactive group and an amino-reactive group to theformation of an antibody or a superantigen carrying the thiol-reactivegroup, and

(ii) reacting the remaining part of the superantigen and the antibodywith an organic reagent containing a thiol group or a protected thiolgroup and an amino-reactive group to the formation of a superantigen oran antibody carrying the thiol group or the protected thiol group,whereupon

(iii) the obtained products from steps (i) and (ii), respectively, arereacted with each other to the formation of a conjugate in which thesuperantigen is linked to the antibody via a disulfide or thioether.

The coupling conditions for each group are known per se as applied toprotein chemistry. The coupling may proceed stepwise or in one step bycreating intermediary functional groups that may be linked to thestarting material by inert spacer arms. In general the conditions underwhich the synthesis and purification/recovering of the conjugate takeplace are always non-denaturing for the proteins involved. This normallymeans aqueous media and a pH-value and temperature within in the rangespH 3-10 and 0°-50° C., respectively. The exact values depend on thegroups to be reacted and the conjugate to be recovered. See more underthe heading Novel reagents.

Novel reagents (developed in connection with the invention)

For the chemical synthesis of conjugates having the linkage --B--, wehave developed a novel heterobifunctional reagent that complies with thegeneral formula II:

    Z.sub.1 RCONHCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m Z.sub.1 '                                                 (II)

m and n have the same meaning as above for formula (I). Z₁ is anHS-reactive electrophilic group, thiol (--SH) or protected thiol (e.g.AcS--), with the provision that a thiol group and a hydroxy group mustnot be bound to one and the same carbon atom in R. Examples ofHS-reactive electrophilic groups are:

(i) halogen that is bound to a saturated carbon atom, preferably in theform of an alfa-halo-alkylcarbonyl (e.g. Z₁ CH₂ CO--);

(ii) activated thiol, preferably a so called reactive disulfide (--SSR₁)that is bound to a saturated carbon atom;

(iii) 3,5-dioxo-1-aza-cyclopent-3-en-1-yl.

For a definition of reactive disulfide see e.g. EP-A-128,885 which isincorporated by reference.

Z₁ ' is an activated carboxy, i.e. an electrophilic group. Examples arecarboxylic acid halides (--COC1, --COBr, and --COI), mixed carboxylicacid anhydrides (--COOOCR₁), reactive esters, such asN-succinimidyloxycarbonyl, --C(═NH)--OR₂, 4-nitrophenylcarboxylate(--CO--OC₆ H₄ NO₂) etc. R₁ and R₂ may be lower alkyl (C₁ -C₆) and R₂also benzyl.

One of the advantages of our novel reagents are that they result inuniform conjugate substances with regard to the integer n in thestructure (OCH₂ CH₂)n, i.e. n is the same for each individual moleculeof a given conjugate substance.

Functional groups reacting with Z₁ ' and Z₁ may be present on nativeantibody active molecules or native superantigens or can be introducedon them. The Z₁ ' and Z₁ terminals can then be reacted selectively withthe appropriate antibody or superantigen in a manner known per se forthese types of groups.

Known techniques encompass chain elongation either starting from ournovel reagent or from the compounds to be conjugated.

Chain elongation utilizing our novel reagent may e.g. result inconjugates in which --B-- is:

    --COR'--S.sub.r --RCONHCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m COY--;                                  (1)

CO binds to NH,

    --COR'--S.sub.r --RCONHCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m CONHNH--R"NHN═--;                   (2)

N═ is usually bound to a sp² -hybridized carbon derived from an oxidizedcarbohydrate structure in an antibody or a superantigen (when being aglycoprotein),

    --CO(CH.sub.2).sub.m O(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 NHCOR'--S.sub.r --(cont.) (cont.)--RCONHCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m COY--;                  (3)

CO-- is bound NH,

    --CO(CH.sub.2).sub.m O(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 NHCOR'--S.sub.r --(cont.) (cont.)--RCONHCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m CONHNH--R"NHN═;     (4)

N═ is bound as above in (2),

R, R' and R" are alkylene selected in the same way as R in formula (I).r has the same meaning as above.

The reagent (Formula II) can be prepared starting from compoundscomplying with formula III:

    NH.sub.2 CH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n O(CH.sub.2).sub.m COOH(III)

m equals an integer 1 or 2. n equals an integer 1-20, such as 2-20 or3-9.

The synthesis of certain compounds complying with formula III with m=1and 2, and n=1-10 have been described before (Jullien et al, TetrahedronLetters 29(1988)3803-06; Houghton and Southby, Synth.Commun.19(18)(1989)3199-3209; and EP-A-410,280 (publ. 20.1.91) and Slama andRando, Carbohydrate Research 88(1981)213-221 and Biochemistry19(1980)4595-4600).

Novel reagents complying with formula II can be synthesized by reactinga compound of formula III with a bifunctional reagent of formulaZ--B'--Z' known per se, where Z=Z₁, B'=R" that is as previously definedfor R and R', and Z'=activated carboxy as defined above. After thereaction the --COOH function is transformed to an activated carboxygroup, e.g. Z₁ '=activated ester, such as N-succinimidyloxycarbonyl,4-nitrophenyloxycarbonyl, 2,4-dinitrophenyloxykarbonyl etc.

The novel compounds of formula (III) and their novel derivatives complywith polyethers having the general formula:

    XCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2 --).sub.n OCH.sub.2 Y(IV)

n is an integer 2-20, preferably 3-20 or 3-9. X is H₂ N- including theprotonated form thereof (⁺ H₃ N--) or substituted H₂ N-- that istransformable to H₂ N--, preferably by hydrolysis or reduction. Examplesare unsubstituted amino (H₂ N--); nitro; amido (carbamido), such aslower acylamido (formylamido, acetylamido . . . hexanoylamido) includingacylamido groups that have electron-withdrawing substituents on thealpha carbon atom of the acyl moiety and then particularly CF₃ CONH--,CH₃ COCH₂ CONH-- etc; phtalimidoyl which possibly is ring substituted;carbamato (particularly R₁ 'OCONH-- and (R₁ 'OCO)(R₂ 'OCO)N--, such asN--(tbutyloxycarbonyl)amino (Boc), N--(benzyloxycarbonyl)amino anddi(N--(benzyloxycarbonyl))amino (Z and diZ, respectively) which possiblyare ring substituted; alkyl amino in which the carbon atom binding tothe nitrogen atom is alpha to an aromatic system, such asN-monobenzylamino and dibenzylamino, N-tritylamino(triphenylmethylamino) etc including analogous groups where the methylcarbon atom (including benzylic carbon atom) atom is replaced with asilicon atom (Si), such as N,N-di(tert-butylsilyl)amino; and4-oxo-1,3,5-triazin-1-yl including such ones that are substituted withlower alkyl in their 3- and/or 5-positions.

Above and henceforth R₁ ' and R₂ ' stand for lower alkyl, particularlysecondary and tertiary alkyl groups, and a methyl group that issubstituted with 1-3 phenyl groups that possibly are ring substituted.Lower alkyl and lower acyl groups have 1-6 carbon atoms.

Y is carboxy (--COOH including --COO⁻⁻) or a group that is transformableto carboxy, preferably by hydrolysis or oxidation. The most importantgroups are the ester groups in which the carbonyl carbon atom or thecorresponding atom in ortho esters binds to the methylene group in theright terminal of formula (I). Examples are alkyl ester groups (--COOR₁'); ortho ester groups (--C(OR₃ ')₃) and reactive ester groups asdefined above. R₃ ' has the same meaning as previously defined for R₁,

Other groups Y are --CHO, --CN, --CONH₂, --CONR₁ 'R₂ ' where R₁ ' and R₂' have the same meaning as previously.

The compound of formula IV may be synthesized from known startingmaterials by combining methods that are known per se. Appropriatesynthetic routes are:

A. Formation of the chain.

B. Transformation of terminal functional groups.

C. Transformation of a symmetric polyether to an unsymmetric ether.

D. Splitting of a bisymmetric chain into two identical fragments.

Convenient starting materials that have the repeating unit --OCH₂ CH₂ --are commercially available. Examples are oligoethylene glycols having2to 6 repeating units. Other suitable compounds with identical terminalgroups are corresponding dicarboxylic acids and diamines.

Convenient starting materials that have different terminal groups areomega-hydroxy monocarboxylic acids in which the terminal groups arespaced apart by a pure polyethyleneoxide bridge. Such compounds havingup to 5 repeating units have been described in the prior art (Nakatsuji,Kawamura and Okahara, Synthesis (1981) p.42).

Pharmaceutical compositions and their manufacture

The pharmaceutical composition of the invention comprises formulationsthat as such are known within the field but now containing our novelconjugate. Thus the compositions may be in the form of a lyophilizedparticulate material, a sterile or aseptically produced solution, atablet, an ampoule etc. Vehicles, such as water (preferably buffered toa physiologically pH-value such as PBS) or other inert solid or liquidmaterial may be present. In general terms the compositions are preparedby the conjugate being mixed with, dissolved in bound to or otherwisecombined with one or more water-insoluble or water-soluble aqueous ornon-aqueous vehicles, if necessary together with suitable additives andadjuvants. It is imperative that the vehicles and conditions shall notadversely affect the activity of the conjugate. Water as such iscomprised within the expression vehicles.

Administration and methods of use.

Normally the conjugates will be sold and administered in predispenseddosages, each one containing an effective amount of the conjugate that,based on the result now presented, is believed to be within the range 10_(/) ug-50 mg. The exact dosage varies from case to case and depend onpatient's weight and age, administration route, type of disease,antibody, superantigen, linkage (--B--) etc.

The administration route is as commonly known within the field, i.e. atarget cell lysing effective amount or a therapeutically effectiveamount of a conjugate according to the invention is contacted with thetarget cells. For the indications specified above this mostly meansparenteral administration, such as injection or infusion (subcutanously,intravenously, intra-arterial, intramuscularly) to a mammal, such as ahuman being. The conjugate may be administered locally or systemicallyto the individual to be treated.

By "target cell lysing effective amount" is contemplated that the amountis effective in activating and directing CTLs to destroy the targetcell.

The invention is defined in the appended claims that are part of thedescription. The invention will now be illustrated by a number ofembodiments that in no way limit the general concept we have discovered.The experimental part presents in Part I the chemical synthesis ofconjugates and in Part II effects of the conjugates prepared in example4 on the activation of T-Cells for lysing target cells.

EXPERIMENTAL PORTION PART 1 PREPARATION OF omega-AMINO-PEG-CARBOXYLICACID

Isopropyl 8-hydroxy-3,6-dioxa-octanoate (1).

Sodium (23 g, 1.0 mole) in form of chips was added in portions todiethylene glycol (500 ml) under nitrogen atmosphere. When the sodiumhad reacted completely, the mixture was cooled to room temperature andbromoacetic acid was added (76 g, 0.5 mole) under stirring. After 18hours at 100° C. the excess of diethylene glycol was distilled off atabout 4 mm Hg. Thereafter isopropyl alcohol (400 ml) and in portionsacetyl chloride (51 g, 0.65 mole) were added. After stirring for 18hours at 65° C. the mixture was cooled to room temperature andneutralized with sodium acetate (3.5 g, 0.15 mole). The mixture wasfiltered and the filtrate evaporated nearly to dryness, whereupon it wasdissolved in water (200 ml). The water phase was extracted with1,1,1-trichloroethane (3×50 ml). The pooled organic phases were washedwith water (20 ml). The product was extracted from the pooled waterphases with dichloromethane (50 ml) that after evaporation gave an oil(55 g).

Isopropyl 11-hydroxy-3,6,9-trioxa-undecanoate (2).

Sodium (23 g, 1.0 mole) in form of chips was added in portions totriethylene glycol (700 ml) under nitrogen atmosphere. When the sodiumhad reacted completely, the mixture was cooled to room temperature andbromoacetic acid was added (76 g, 0.5 mole) under stirring. After 18hours at 100° C. the excess of diethylene glycol was distilled off atabout 4 mm Hg. Thereafter isopropyl alcohol (400 ml) and in portionsacetyl chloride (51 g, 0.65 mole) were added. After stirring for 18hours at 65° C. the mixture was cooled to room temperature andneutralized with sodium acetate (3.5 g, 0.15 mole). The mixture wasfiltered and the filtrate evaporated nearly to dryness, whereupon it wasdissolved in water (200 ml). The water phase was extracted with1,1,1-trichloroethane (3×50ml). The pooled organic phases were washedwith water (20 ml). The product was extracted from the pooled waterphases with dichloromethane (50 ml) that after evaporation gave an oil.

¹ H-n.m.r.(CDCl₃); 1.26(d,6H);3.07(s,2H);3.6-3.8(m,12H);4.11(s,2H);5.09(m,1 H)

8-(N-phtalimidoyl)-3,6-dioxa-octanol (3).

8-Chloro-3,6-dioxa-octanol (365 g, 2.2 mole, prepared from fromtriethylene glycol and SOCl₂) was dissolved in dimethyl formamide (400ml) and potassium phtalimide (370 g, 2.0 mole) was added under stirring.After stirring for 18 hours at 110° C. dimethyl formamide was distilledoff at reduced pressure. The residue was suspended in toluene (1.5 l) at40°-50° C. and potassium chloride was filtrated off. The productcrystallizes at cooling (-10° C.). A second fraction is available fromthe mother liquor by concentrating it and repeating the crystallizationprocedure.

¹ H-n.m.r.(CDCl₃); 2.90(s,₁H);3.51-3.58(m,2H);3.60-3.68(m,6H);3.73-3.78(t,2H);3.89-3.94(t,2H);7.70-7.89(m,4H).

Isopropyl 17-(N-phtalimidoyl)-3,6,9,12,15-pentaoxa-heptadecanoate (4).

A solution of pyridine (2.8 ml, 35 mmole) in dichloromethane (30 ml) wasadded dropwise under stirring at about -5° C. to a solution of8-(N-phtalimidoyl)-3,6-dioxa-octanol (3) (8.5 g, 36 mmole) andtrifluoromethanesulfonic acid anhydride (10.2 g, 36 mmole) indichloromethane. After about 30 minutes the organic phase was washedwith 0.5M hydrochloric acid and water. After drying (Na₂ SO₄) andfiltration isopropyl 8-hydroxy-3,6-dioxa-octanoate (1) (12 g, 48 mmole)and Na₂ PO₄ (6.5, 46 mmole) were added, and the mixture was vigorouslystirred for 20 hours at room temperature. The reaction mixture wasfiltrated and the filtrate evaporated. The residue was partitionedbetween 1,1,1-trichloroethane and water. Evaporation of the organicphase resulted in an oil (13 g).

¹ H-n.m.r.(CDCl₃);1.26(d,6H);3.58-3.76(m,18H);3.90(t,2H);4.11(s,2H);5.09(m,1H);7.70-7.89(m,4H).

17-(N-phtalimidoyl)-3,6,9,12,15-pentaoxa-heptadecanoic acid (5).

Isopropyl 17-(N-phtalimidoyl)-3,6,9,12,15-pentaoxa-heptadecanoate (4)(13 g) was dissolved in tetrahydrofuran (50 ml) and hydrochloric acid(conc., 50 ml)). After 16 hours at room temperature the solution wasdiluted with water (200 ml) and tetrahydrofuran was removed at reducedpressure. The water phase was washed with toluene (1×) and extractedwith dichloromethane (2×). Drying (Na₂ SO₄) and evaporation of theorganic phase resulted in the product in form of an oil (8.5 g)

¹ H-n.m.r.(CDCl₃): 3.57-3.76 (m,18H);3.91(t,2H);4.11(s,2H);4.8(br,2H);7.65-7.90(m,4H)

Isopropyl 17-amino-3,6,9,12,15-pentaoxa-heptadecanoate (6)

17-(N-phtalimidoyl)-3,6,9,12,15-pentaoxa-heptadecanoic acid (5) (8.5 g)was dissolved in 150 ml ethanol and 3 ml hydrazine hydrate. The solutionwas stirred at room temperature for 16 hours, whereupon HC1 (100 ml, 3M)was added and the solution was then refluxed for 3 hours. After coolingto room temperature and filtration, pH was adjusted (pH 9, NaOH) and thefiltrate was evaporated almost to dryness. Water was added andre-evaporation almost to dryness was carried out, whereupon the pH ofthe solution was adjusted (pH 4, HC1) followed by evaporation todryness. The product was treated with isopropanol (100 ml) and acetylchloride (2 ml) at room temperature during the night and evaporated. Theresidue was collected in water and extracted into dichloromethane at analkaline pH (7-11). Evaporation resulted in the product (3.3 g).

¹ H-n.m.r.(CH₃ OD): 1.26(d,6H);3.17(t,2H);3.65-3.80(m,18H);4.16(s,2H);5.07(m,1H)

FORMULAE OF SYNTHESIZED AMINO-PEG-CARBOXYLIC ACIDS

    H--(OCH.sub.2 CH.sub.2).sub.n CH.sub.2 CO--OCH(CH.sub.3).sub.2

Compound 1:n=1 Compound 2:n=1

    PhtN--CH.sub.2 CH.sub.2 - (OCH.sub.2 CH.sub.2).sub.2 OH

Compound 3, PhtN--=N-phtalimidoyl

    PhtN--CH.sub.2 CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.4 CH.sub.2 CO--OCH(CH.sub.3).sub.2

Compound 4, PhtN--=N-phtalimidoyl

    PhtN--CH.sub.2 CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.4 CH.sub.2 CO--OH

Compound 5

    H.sub.2 N--CH.sub.2 CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.4 CH.sub.2 CO--OH

Compound 6 PREPARATION OF BIFUNCTIONAL REAGENTS AND COUPLING PRODUCTSStructural formulae are set forth on a separate page. Example 1

Preparation of N-hydroxysuccinimide ester of17-iodoacetylamino-3,6,9,12,15 pentaoxaheptadecanoic acid

A. Preparation of 17-iodoacetylamino -3,6,9,12,15-pentaoxaheptadecanoicacid (A)

Isopropyl 17-amino-3,6,9,12,15-pentaoxahepta-decanoate (see part I ofthe experimental part) (1.1 g, 3.2 mmole) was dissolved in 3 ml of 1Msodium hydroxide solution and left at room temperature for 30 min. 1.5ml of 6M hydrochloric acid was added and the mixture was evaporated todryness. The residue was taken up in dichloromethane and filtered togive 545 mg of 17-amino-3,6,9,12,15-pentaoxa-heptadecanoic acid afterevaporation of the solvent. 460 mg (1,39 mmoles) of this compound weredissolved in 10 ml of borate buffer pH 8.4. The solution was deaeratedwith nitrogen gas. A solution of 432 mg (1.52 mmoles) of N-succinimidyl2-iodoacetate in 5 ml of dioxane was added dropwise during 1 min pH waskept at 8.4 by addition of 5M NaOH. The reaction solution was stirredfor 15 min during inlet of nitrogen gas. According to thin layerchromatography (eluent: CH₂ Cl₂ --MeOH 60:35) the reaction was completedin some few minutes. After 15 min the pH of the reaction solution wasadjusted to 3 and the solution was frozen and lyophilized. The reactionmixture was fractionated on a reversed phase column PEP--RPC HR 30/26(Pharmacia Biosystems AB) using a gradient of 0-13% acetoniIrile with0.1% tri-fluoroacetic acid followed by isocratic separation at 13%acetonitrile, 0.1% TFA. Fractions from the desired peak were pooled andlyophilized giving 351 mg of17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoic acid (A). Yield:76%.

The structure of the product was established by the aid of its NMRspectrum. ¹ H NMR spectrum (D₂ 0) expressed as δ-values: ##STR1##

B. Preparation of N-hydroxysuccinimide ester of17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid (B)

Hydroxysuccinimide (4.5 mg, 39 μmole) was weighed in the reaction vial.17-Iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid (A) (18.3 mg,39 μmole) was dissolved in 0.55 ml dried dioxane and added to thereaction vial. The vial was deareated with nitrogen gas and then asolution of 8.0 mg (39 μmole) dicyclohexylcarbodiimide in 0.15 ml ofdried dioxane was added dropwise to the reaction vial. The vial wasfilled with nitrogen gas, closed and placed in the dark. The reactionsolution was stirred for 3.5 h. The precipitate formed was removed byfiltration. The percentage formed product B in the filtrate wasdetermined by NMR-analysis to be 89%.

Example 2

Preparation of(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)-immunoglobulin(C)

A. Monoclonal antibody Mab C215

A monoclonal antibody of immunoglobulin class IgG2a (Mab C215) (34 mg,0.218 μmole) dissolved in 17.7 ml of 0.1M borate buffer pH 8.1containing 0.9% sodium chloride was added to a reaction vial. 146 μl ofa dioxane solution containing 3.6 mg (6.4 μmole) of N-hydroxysuccinimideester of 17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid (B)was injected into the buffer solution and the reaction was completedduring stirring for 25 min. at room temperture. The reaction vial wascovered with foil to exclude light. Excess of reagent B was removed byfractionation on a Sephadex G 25 K 26/40 column using 0.1M phosphatebuffer pH 7.5 containing 0.9% sodium chloride as eluent. Fractionscontaining the desired product C were pooled. The solution (22 ml) wasconcentrated in an Amicon cell through a YM 30 filter to 8 ml. Theconcentration and degree of substitution were determined with amino acidanalysis to be 4.7 mg/ml and 18 spacer per Mab C215 respectively.

B. Monoclonal antibody Mab C242

A monoclonal antibody (Mab C242) of the immunoglobulin class IgG 1 wasreacted with 15, 20 and 22 times molar excess of N-hydroxysuccinimideester of 17-iodoacetyl-amino-3,6,9,12,15-pentaoxaheptadecanoic acid (B)respectively according to the procedure described in example 2.A givingnona, dodeca andtetradeca(17-iodoacetylamino)-3,6,9,12,15-pentaoxaheptadecanoylamino)-MabC242. (C)

C. Monoclonal antibody Mab C

A monoclonal antibody (Mab C) of the immunoglobulin class IgG 2a wasreacted with 14 and 18 times molar excess of N-hydroxysuccinimide esterof 17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoic acid (B)respectively according to the procedure described in example 2A givingtetra andhepta(17-iodoacetylamino-3,6,9,12,15-pentaoxa-heptadecanoylamino)-Mab C.(C)

Example 3

Preparation of 2-mercaptopropionylamino-Eu³ -labelled-staphylococcalenterotoxin A (SEA)

A. Preparation of Eu³⁺ labelled SEA (D)

SEA (freezed dried product from Toxin Technology Inc.) (2 mg, 72 nmole)was dissolved in 722 μl milli-Q water and added to a 15 ml polypropylenetube. 100 μl of 0.1M borate buffer pH 8.6 was added and then 2160 nmolesof Eu³⁺ -chelate reagents (Pharmacia Wallac Oy) in 178 μl of milli-Q.The reaction was completed at room temperature over night. Excessreagent was removed by fractionation of the reaction solution on aSephadex G 25 PD 10 column (Pharmacia Biosystems AB) using 0.1Mphosphate buffer pH 8.0 as eluent. Fractions with the desired product Dwere pooled. The solution (3 ml) was concentrated in an Amicon cellthrough an YM5 filter to a volume of 0.8 ml. The concentration wasdetermined with amino acid analysis to be 1.7 mg/ml. The degree ofsubstitution was determined by comparing with a EuCl₃ standard solutionto be 0.8 Eu³⁺ per SEA.

B1. Preparation of 3-(2-pyridyldithio)propionylamino Eu³⁺ labelled SEA(E) and 3-mercaptopropionylamino Eu³⁺ labelled SEA (F)

Eu³⁺ -SEA (1.24 mg, 44.8 nmoles) in 0.75 ml of 0.1M phosphate buffer pH8.0 was added to a 15 ml polypropylene tube. 35 μl (180 nmole) of asolution of 1.6 mg of N-succinimidyl 3-(2-pyridyldithio)-propionate in 1ml of ethanol was added to the tube and the reaction solution wasstirred for 30 min at room temperature. The obtained product E was notisolated before being reduced to product F.

To the reaction solution from above were added 20 μl of 0.2M Eu³⁺-citrate solution and 50 μl of 2M acetic acid to adjust the pH to 5.Thereafter a solution of 3.1 mg of dithiotreitol (Merck) in 0.1 ml of0.9% sodium chloride was added and the reaction solution was stirred for20 min at room temperature. Thereafter the total volume was adjusted to1 ml by addition of 50 μl of 0.9% sodium chloride solution. The reactionsolution (1 ml) was placed on a Sephadex G25 NAP-10 column (PharmaciaBiosystems AB) and desired product F was eluted by addition of 1.5 ml of0.1M phosphate buffer pH 7.5 containing 0.9% sodium chloride. The elutedproduct F was collected in a 15 ml polypropylene tube and immediatelyused in the synthesis of product G to avoid reoxidation to a disulfidecompound.

B2. Preparation of 2-mercaptopropionylaminostaphylococcal enterotoxin A(SEA) (F2)

Native SEA (freeze dried product from Toxin Technology Inc) orrecombinant prepared SEA (rSEA) was reacted with 2 times molar excess ofN-succinimidyl 3-(2-pyridyldithio)-propionate according to the proceduredescribed in example 3B1.

The degree of substitution was determined with UV-analysis according toCarlsson et al (Biochem. J. 173(1978)723-737) to be 1.9mercaptopropionyl group per SEA.

Example 4

Preparation of the SEA-monoclonal antibody conjugate (G1 och G2)

A. Conjugates between Eu³⁺ -SEA and Mab C215 (G1)

To the solution of 4-mercaptopropionylamino Eu³⁺ labelled SEA (F)described in example 3B was added 1.2 ml of a solution ofoctadeca(17-iodoactylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC215 (C) (4 mg) in 0.1M phosphate buffer pH 7.5 containing 0.9% sodiumchloride. The reaction was completed by standing at room temperatureover night. Unreacted iodinealkyl groups were then blocked by additionof 5 μl (1.2 μmole) of a solution of 20 μl mercaptoethanol in 1 ml ofwater. The reaction solution was left for 4 h at room temperature andthen filtrated. The filtrate was then fractionated on a Superose 12 HR16/50 column (Pharmacia Biosystems AB) using as eluent 0.002M phosphatebuffer pH 7.5 containing 0.9% sodium chloride. Fractions with thedesired product G were pooled and analysed. The protein content was 0.22mg/ml determined by amino acid analysis. The degree of substitution wasone SEA per IgG determined by Eu³⁺ determination. The product was alsostudied for immunostimulating properties and antibody binding capacity.

By increasing the amount of compound (F) in relation to compound (C)higher degree of substitution was obtained.

B. Conjugates between rSEA and Mab C215 (G2)

Octadeca (17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC215 (C) was reacted with 1.8 times molar excess of2-mercaptopropionylamino-rSEA (F2) according to the procedure describedin example 4A. The composition of the conjugate was analysed by sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) onPhast-Gel™ gradient 4-15 and the bonds were scanned with Phast IMAGE(Pharmacia Biosystems AB). The conjugate obtained was composed of 6% MabC215 with three SEA, 15% with two SEA, 28% with one SEA and 51%unsubstituted Mab C215.

In another experiment 2.7 times molar excess of F2 was used giving aconjugate with the composition 15% Mab C215 with three SEA, 25% with twoSEA, 34% with one SEA and 26% of unsubstituted Mab C215.

C. Conjugates between rSEA and Mab C242

C1.Tetradeca(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC242 (C) was reacted with 3.2 times molar excess of2-mercaptopropionylamino-rSEA (F2) according to the procedure describedin example 4A. The composition of the conjugate was analysed asdescribed in example 4 B and was found to be 4% Mab C242 with four SEA,12% with three SEA, 28% with two SEA, 36% with one SEA and 20% ofunsubstituted Mab C242.

The same reaction was run but the reaction product was treated 4 h with0.2M hydroxylamine before column fractionation to remove unstable bondsbetween Mab C242 and the spacer and between SEA and themercaptopropionyl group. The conjugate formed had the composition 1% MabC242 with four SEA, 12% with three SEA, 27% with two SEA, 36% with oneSEA and 24% of unsubstituted Mab.

C2. Dodeca(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC242 (C) was reacted with 3 times molar excess of2-mercaptopropionylamino-rSEA (F2) according to the procedure describedin example 4A. The conjugate obtained had the composition 6% Mab C242with three SEA, 26% with two SEA, 36% with one SEA and 31% unsubstitutedMab C242.

C3. Nona(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC242 (C) was reacted with 3 times molar excess of2-mercaptopropionylamino-rSEA (F2) according to the procedure describedin example 4A.

The conjugate obtained had the composition 13% Mab C242 with two SEA,39% with one SEA and 46% unsubstituted Mab C242.

D. Conjugates between rSEA and Mab C

D1. Hepta(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC was reacted with 5.4 times molar excess of2-mercaptopropionylamino-rSEA (F2) according to the procedure describedin example 4A. The composition of the conjugate was analysed asdescribed in example 4B and was found to be 15% with four SEA, 24% withthree SEA, 29% with two SEA, 19% with one SEA, 3% unsubstituted Mab Cand 10% in a dimeric form.

The same reaction was run with 0.2M hydroxylamine present to removeunstable bonds between Mab C and spacer and between SEA and themercaptopropionyl group. The conjugate formed had the followingcomposition 11% Mab C with three SEA, 24% with two SEA, 30% with oneSEA, 18% of unsubstituted Mab C and 17% in a dimeric form.

D2. Tetra(17-iodoacetylamino)-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC was reacted with 5.7 times molar excess of2-mercaptopropionylamino-rSEA (F2) according to the procedure describedin example 4B. The conjugate had the following compositon 8% Mab C2 withfour SEA, 18% with three SEA, 30% with two SEA, 26% with one SEA, 5% ofunsubstituted Mab C and 12% in a dimeric form.

Example 5

Preparation of17-(3-mercaptopropionylamino)-3,6,9,12,15-pentaoxaheptadecanoylamino!-rSEA(I)

A. Preparation of 17-3-(2-pyridyldithio)propionylaminol-!3,6,9,12,15-pentaoxaheptadecanoylamino)-rSEA(H)

A solution of N-hydroxysuccinimide ester of 17-3-(2-pyridyldithio)propionylamino!-3,6,9,12,15-pentaoxaheptadecanoicacid (K) (0.53 mg (896 nmoles) in 43 μl of dioxane) was injected into asolution of 3.67 mg (128 nmoles) of rSEA in 1 ml of 0.1M phosphatebuffer pH 7.5 containing 0.9% of sodium chloride. The reaction wascompleted in 30 min at room temperature. 100 μl was taken foranalysation of degree of substitution. The rest was stored frozen untilit was reduced to product I.

The degree of substitution was determined by desalting 100 μl of thereaction solution on a Sephadex G50 NICK column (Pharmacia BiosystemsAB) and analysing the eluate with UV-spectroscopy according to Carlssonet al (Biochem. J. 173(1978)723-737). 2.7 spacers were coupled to rSEA.

B. Preparation of 17-(3-mercaptopropionylamino)-36,9,12,15-pentaoxaheptadecanoylamino!-rSEA (I)

The pH of the reaction solution with product H (0,9 ml) was adjustedwith 2 HC1 to pH 4.4 and 2.9 mg of dithiotreitol dissolved in 75 μl 0.9%sodium chloride was added. The reduction was completed in 30 min. Thereaction solution was added to a Sephadex G25 NAP 10 column (PharmaciaBiosystems AB) and eluted with 1.5 ml of 0.1M phosphate buffer pH 7.5with 0.9% NaCl and immediately used in the synthesis of product J inexample 11.

Example 6

Preparation of SEA-monoclonal antibody conjugate J with double spacer

Dodeca(17-iodoacetylamino-3,6,9,12,15-pentaoxaheptadecanoylamino)MabC242 (C) (4.2 mg, 27 nmoles in 1.0 ml of 0.1M phosphate buffer pH 7.5with 0.9% NaCl) was reacted during 43 h with17-(3-mercaptopropionylamino)-3,6,9,12,15-pentaoxaheptadecanoylamino!-rSEA(I) (1.17 mg, 42 nmoles in 1 ml of the above buffer) in the dark innitrogen atmosphere. Thereafter 1.14 μmole of mercaptoethanol was added.After another 1 h the reaction solution was fractionated on a Superdex200 HR 16/65 column. The product was eluated with 2 mM phosphate bufferpH 7.5 with 0.9% NaCl. Fractions with the desired product J were pooledand analysed as described in example 4B. The conjugate was composed of9% Mab C242 with two SEA, 25% with one SEA and 66% of unsubstituted MabC242. ##STR2##

EXPERIMENTAL PART II Effects of superantigen-antibody conjugates oncells

The bacterial toxin used in the following experiments was Staphylococcusenterotoxin A (SEA) obtained from Toxin Technologies (WI; USA) orproduced as a recombinant protein from E. Coli.

The antibodies were C215, C242 and Thy-1.2 mAbs. C215 is an lgG2a mAbraised against human colon carcinoma cell line and reacts with a 37kDprotein antigen on several human colon cell lines. References to thesemAbs have been given above. The conjugates were prepared as described inthe preceding part.

Before the priority date studies had only been performed with Eu₃₊labelled SEA-C215 mAb conjugates. During the priority year the resultshave been verified with unlabelled SEA-215, SEA-242 and SEA-Thy-1.2 mAbconjugates. The results now incorporated refer to unlabelled conjugates.

To determine the cytotoxicity mediated by the SEA-C215 mAb conjugate andunconjugated SEA and C215 mAb against colon carcinoma cells lacking MHCClass II or expressing low but undetectable amounts of MHC Class II, weemployed various human SEA expanded T cell lines as effector cells and apanel of colon carcinoma cells and MHC Class II⁺ Raji cells as targetcells. The colon carcinoma cell lines Colo205, SW620 and WiDr, alllacked expression of MHC Class II, as determined by staining with mAbsagainst HLA-DR, HLA-DP and HLA-DQ and FACS analysis. The SEA expanded Tcell lines were established from peripheral blood by weeklyrestimulations with mitomycin C treated MHC Class II⁺ BSM lymphoma cellsprecoated with SEA in the presence of recombinant IL-2 (20 units/ml).These T cell lines were strongly cytotoxic towards Raji or BSM cellscoated with SEA but not to uncoated cells or cells coated withstaphylococcal enterotoxin B (SEB). This SEA induced killing isdependent on interaction of SEA with MHC Class II on the target cell asdetermined by the use of blocking HLA-DR antibodies, MHC Class II⁻ Rajimutant cells and HLA-DR transfected L-cells (Dohlsten et al., Immunology71 (1990) 96-100. These T cell lines could be activated to kill C215⁺MHC Class II⁻ colon carcinoma cells by the C215-SEA conjugate. Incontrast unconjugated SEA and C215 mAb were unable to induce more thanmarginal T cell killing against the C215⁺ MHC Class II⁻ colon carcinomacells. The staphylococcal enterotoxin antibody conjugate dependentcell-mediated cytotoxicity was dependent on binding of the SEA-C215 mAbconjugate to the C215⁺ tumor cells. The specificity in this binding wasdemonstrated by the fact that excess of unconjugated C215 mAb but notthe irrelevant C242 and w6/32 mAbs inhibited the lysis of the coloncarcinoma cells. CD4⁺ and CD8⁺ T cells demonstrated killing of SEA-C215treated C215⁺ colon carcinoma cells, but did not lyse SEA treated cells.The interaction of T cells with SEA-C215 mAb conjugate bound to MHCClass II⁻ tumor cell seems to involve interaction with specific V-betaTCR sequences in a similar manner as earlier demonstrated for SEAinduced killing of MHC Class II⁺ cells. This was indicated by theinteraction of an SEA specific but not an autologous SEB specific T cellline with the C215-SEA conjugate. C242 mAb and Thy-1.2 mAb conjugatesdemonstrate activity in analogy with the C215 mAb conjugate.

Chromium labelling and incubation of the target cells with SEA

0.75×10⁶ target cells and 150 μCi ⁵¹ chromium (Amersham Corp., ArlingtonHights, England) were incubated for 45 minutes at 37° C. in a volume of100 μl. The cells were kept in complete medium containing RPMI-1640medium (Gibco, Paisley, GBR) supplemented with 2.8% (v/v) 7.5% NaHCO₃,1% sodium pyrovate, 2% 200 mM L-glutamine, 1% 1M Hepes, 1% 10 mg/mlgentamicin and 10% fetal calf serum (FCS, Gibco, Paisley, GBR). Afterthe incubation the cells were washed once in complete medium without FCSand incubated 60 minutes at 37° C. and washed and resuspended incomplete medium containing 10% FCS. 5×10³ target cells were added toeach well of U-bottom 96-well microtiter plates (Costar, Cambridge,USA).

Cytotoxicity assay

The effector cells were added to the wells at various effector/targetcell ratios. The final volume in each well was 200 μl. Each test wasdone in triplicate. The plates were incubated 4 hours at 37° C. afterwhich the released chromium was harvested. The amount 51Cr wasdetermined in a gamma-counter (Cobra Auto-gamma, Packard). Thepercentage cytotoxicity was computed by the formula %cytotoxicity=(X-M)/(T-M)*100, where X is the chromium release as cpmobtained in the test sample, M is the spontaneous chromium release oftarget cells incubated with medium, and T is the total chromium releaseobtained by incubating the target cells with 1% sodium dodecyl sulfate.

RESULTS

SEA-C242, SEA-C215 and SEA-anti-Thy-1.2 mAb conjugates bind to cellsexpressing the relevant epitopes of the mAbs, respectively, and to MHCClass II⁺ cells. Unconjugated SEA on the other hand only binds to MHCClass II⁺ cells. Unconjugated C215, C242 and Thy-1.2 mAbs bind to therelevant cells but not to Raji cells. (Table 1)

Human T cell lines lysed the MHC Class II⁻ SW620, Colo205 and WiDr cellsin the presence of SEA-C215 mAb conjugate but not in the presence ofunconjugated SEA and C215 mAb (FIG. 1). The lysis of colon carcinomacells was seen at 10-100 ng/ml of SEA-C215 mAb conjugate. High levels oflysis at various effector to target ratios were seen with SEA-215 mAbconjugate against SW620 (FIG. 1). In contrast, unconjugated SEA or C215mAb mediated no cytotoxicity against SW620 cells at all tested effectorto target ratios. This indicates that the capacity to lyse MHC Class II⁻Colo205 cells is restricted to the conjugate and cannot be induced byunconjugated SEA and C215 mAb. SEA and SEA-C215 mAb conjugate but notC215 mAb mediated T cell killing of MHC Class II⁺ Raji cells and ofinterferon treated MHC Class II⁺ Colo205 cells (FIG. 1).

In order to demonstrate that the SEA-C215 mAb conjugate mediated lysisinvolved specific binding of the conjugate to the C215 mAb molecule onthe target cells, we performed blocking studies with excess ofunconjugated C215 mAb and mAb C242, which bind to an irrelevant antigenon the colon carcinoma cells (in regard to C215 mAb binding). Additionof mAb C215 strongly blocked cytotoxicity, whereas the C242 mAb had noinfluence (FIG. 2). Similarly lysis by a SEA-C242 mAb conjugate wasspecifically blocked by excess of unconjugated C242 mAb but not C215mAb.

The capacity of SEA-C215 mAb conjugate to induce T cell dependent lysisof MHC Class II SW620 colon carcinoma cells was seen in both CD4⁺ andCD8⁺ T cell populations (Table 2). SEA did not activate any of these Tcell subsets to mediate killing of SW620 cells but induced lysis of MHCClass II⁺ Raji cells (Table 2).

The SEA-C215 mAb conjugate induced lysis of SW620 and Raji cells by aSEA expanded T cell line, but not by a SEB expanded T cell line (FIG.3). The specificity of the SEA and SEB lines is indicated by theirselective response to SEA and SEB, respectively, when exposed to Rajicells (FIG. 4). This indicates that the SEA-C215 mAb conjugate retainssimilar V-beta TCR specificity as for unconjugated SEA.

Legend to figures

FIG. 1. The SEA-C215 mAb conjugate directs CTLs against MHC class II⁻colon carcinoma cells. Upper left panel demonstrates the effect of SEAresponsive CTLs against SW620 cells at various effector to target ratiosin the absence (-) or presence of SEA-C215 mAb conjugate, SEA, C215 anda mixture of C215 and SEA (C215+SEA) at a concentration of 1 μg/ml ofeach additive. The other panels demonstrates the capacity of SEA--C215mAb conjugate, and SEA to target SEA responsive CTLs against theC215+MHC class II⁻ colon carcinoma cell lines SW620, Colo205 and WiDr,MHC class II⁺ C215⁺ interferon treated Colo205 cells and C215- MHC classII+Raji cells. Effector to target ratio was 30:1. Addition ofunconjugated C215 mAb, at several concentrations, did not induce any CTLtargeting against these cell lines. FACS analysis on SW620 cells,Colo205 and WiDr cells using mAbs against HLA--DR, --DP, --DQ failed todetect any surface MHC class II expression, whereas abundant expressionof HLA--DR, --DP and --DQ was detected on Raji cells and HLA--DR and--DP on interferon treated Colo205 cells. Colo205 cells were treatedwith 1000 units/ml of recombinant interferon-gamma for 48 hours prior touse in the CTL assay.

FIG. 2. SEA-C215 mAb conjugate and SEA-C242 mAb conjugate induced CTLtargeting against colon carcinoma cells depends on the antigenselectivity of the mAb. Lysis of Colo205 cells by a SEA responsive CTLline in the presence of SEA-C215 mAb and SEA-C242 mAb conjugate (3μg/ml) is blocked by addition of unconjugated C215 and C242 mAbs (30μg/ml), respectively. The unconjugated mAbs or control medium (-) wereadded to the target cells 10 minutes prior to the conjugates.

FIG. 3. Lysis of SEA-C215 mAb conjugate coated colon carcinoma cells ismediated by SEA but not SEB responding CTLs. Autologous SEA and SEBselective T cell lines were used at an effector to target ratio of 10:1against SW620 and Raji target cells in the absence (control) or presenceof SEA-C215 mAb conjugate, a mixture of unconjugated C215 mAb and SEA(C215+SEA) and unconjugated C215 mAb and SEB (C215+SEB) at aconcentration of 1 μg/ml of each additive.

FIG. 4. Cytotoxicity induced by the SEA-C242 mAb conjugate andSEA-Anti-Thy-1.2 mAb conjugate against their target cells (Colo205tumour cells and EL-4 tumour cells, respectively).

                  TABLE 1    ______________________________________    SEA-C215 mAb conjugate bind to    C215.sup.+  colon carcinoma cells and MHC Class II.sup.+  Raji cells    Reagent          Cell     Facs analysis    ______________________________________    SEA-C215 mAb     Colo205  Pos                     Raji     Pos    C215 mAb         Colo205  Pos                     Raji     Neg    SEA-C242 mAb     Colo205  Pos                     Raji     Pos    C242 mAb         Colo205  Pos                     Raji     Neg    SEA-anti-Thy-1.2 mAb                     EL-4     Pos    anti-Thy-1.2 mAb EL-4     Pos    SEA              Colo205  Neg                     Raji     Pos    control          Colo205  Neg                     Raji     Neg                     EL-4     Neg    ______________________________________

Cells were incubated with the various additives of control (PBS-BSA) for30 minutes on ice, washed and processed as described below. The stainingof C215 mAb and C242 mAb bound to Colo205 cells and anti-Thy-1.2 boundto EL-4 cells was detected using FITC labelled rabbit anti mouse 1 g.The staining of SEA to Raji cells was detected using a rabbit anti-SEAsera followed by a FITC-swine anti rabbit 1 g. The staining of SEA-C215mAb conjugate to Colo 205 and Raji cells was detected utilizing theabove described procedures for C215 mAb and SEA. FACS analysis wasperformed on a FACS star plus from Becton and Dickinson. Staining withsecond and third steps only was utilized to define the background.

                  TABLE 2    ______________________________________    CD4.sup.+  and CD8.sup.+  CTLs lyse colon carcinoma cells    presenting the C215-SEA conjugate.                % cytotoxicity    Effector.sup.A)              Target  control     SEA  C215-SEA    ______________________________________    CD4.sup.+ SW620   2            5   50    CD4.sup.+ Raji    0           41   43    CD8.sup.+ SW620   0            1   23    CD8.sup.+ Raji    2           72   68    ______________________________________     .sup.A) The CTLs (SEA3) were used at effector to target ratios of 30:1 in     the absence (control) or presence of SEA and C215SEA at 1 μg/ml.

We claim:
 1. A soluble antibody conjugate comprising a superantigencovalently linked to an antibody, wherein said antibody is specific fora cell surface structure on a cell, and said superantigen is recognizedby T-Cells and capable of activating cytotoxic T-cells(CTL's).
 2. Aconjugate according to claim 1, wherein said antibody is monoclonal. 3.A conjugate according to claim 2, wherein said cell is associated withdisease selected from the group consisting of cancer, autoimmunity,parasitic infestation, and microbial infections.
 4. A conjugateaccording to claim 3, wherein said disease is cancer.
 5. A conjugateaccording to claim 4, wherein said cell is a colon carcinoma.
 6. Aconjugate according to claim 3, wherein said surface structure is a C242epitope.
 7. A conjugate according to claim 3, wherein there are 1 to 5superantigen moieties per antibody moieties.
 8. A conjugate according toclaim 3, wherein the superantigen is of bacterial origin.
 9. A conjugateaccording to claim 3, wherein the superantigen is a staphylococcalenterotoxin.
 10. A conjugate according to claim 9, wherein thesuperantigen is SEA.
 11. A conjugate according to claim 2, wherein thesurface structure is a C242 epitope and the superantigen is SEA.
 12. Aconjugate according to claim 2, wherein the superantigen and antibodyare held together through a covalent organic linkage structure B whichcomprises at least one amide structure.
 13. A conjugate according toclaim 12, wherein the linkage B comprises the structure --S_(r) RCONHCH₂CH₂ (OCH₂ CH₂)_(n) O(CH₂)Coy--(I) whereinr is an integer 1 or 2 R isalkylene having 1-4 carbon atoms; n is an integer from 1-20; m is aninteger 1 or 2; and Y is --NH--, --NHNH-- or --NHN═CH--.
 14. A methodfor the lysis of a target cell, by assistance of T-cells, wherein thetarget cells are contacted with a target cell lysis effective amount ofa soluble antibody conjugate comprising a monoclonal antibody or anantibody fragment linked to a superantigen; wherein said monoclonalantibody or antibody fragment is specific for a cell surface structureon said target cell, and said superantigen is recognized by T-cells andcapable of activating cytotoxic T-cells (CTL's).
 15. A method for thetreatment of a mammal, including a human individual, suffering from adisease selected from the group consisting of cancer; autoimmunity; aninfection caused by a virus, a bacteria or fungi; and an infestationcaused by a parasite, said method comprising administering to saidmammal a target cell lysis effective amount of a conjugate according toclaim
 1. 16. A method according to claim 14, wherein said target cell isassociated with disease selected from the group consisting of cancer,autoimmunity, parasitic infestation, and microbial infections.
 17. Amethod according to claim 16, wherein said disease is cancer.
 18. Amethod according to claim 17, wherein said target cell is a coloncarcinoma cell.
 19. A method according to claim 16, wherein said surfacestructure is a C242 epitope.
 20. A method according to claim 16, whereinthere are 1 to 5 superantigen moieties per antibody moieties.
 21. Amethod according to claim 16, wherein the superantigen is of bacterialorigin.
 22. A method according to claim 16, wherein the superantigen isa staphylococcal enterotoxin.
 23. A method according to claim 22,wherein the superantigen is SEA.
 24. A method according to claim 16,wherein the surface structure is a C242 epitope and the superantigen isSEA.
 25. A method according to claim 16, wherein the superantigen andantibody are held together through a covalent organic linkage structureB which comprises at least one amide structure.
 26. A method accordingto claim 25, wherein the linkage B comprises the structure --S_(r)RCONHCH₂ CH₂ (OCH₂ CH₂ )_(n) O (CH₂)_(m) COY--(I) whereinr is an integer1 or 2 R is alkylene having 1-4 carbon atoms; n is an integer from 1-20;m is an integer 1 or 2; and Y is --NH--, --NHNH-- or --NHN═CH--.
 27. Amethod according to claim 15, wherein said disease is cancer.
 28. Amethod according to claim 15, wherein said target cell is a coloncarcinoma cell.
 29. A method according to claim 15, wherein said surfacestructure is a C242 epitope.
 30. A method according to claim 15, whereinthere are 1 to 5 superantigen moieties per antibody moieties.
 31. Amethod according to claim 15, wherein the superantigen is of bacterialorigin.
 32. A method according to claim 15, wherein the superantigen isa staphylococcal enterotoxin.
 33. A method according to claim 32,wherein the superantigen is SEA.
 34. A method according to claim 15,wherein the surface structure is a C242 epitope and the superantigen isSEA.
 35. A method according to claim 15, wherein the superantigen andantibody are held together through a covalent organic linkage structureB which comprises at least one amide structure.
 36. A method accordingto claim 35, wherein the linkage B comprises the structure --S_(r)RCONHCH₂ CH₂ (OCH₂ CH₂)_(n) O (CH₂)_(m) COY--(I) whereinr is an integer1 or 2 R is alkylene having 1-4 carbon atoms; n is an integer from 1-20;m is an integer 1 or 2; and Y is --NH--, --NHNH-- or --NHN═CH--.
 37. Aconjugate according to claim 1, wherein said antibody comprises anactive antibody fragment.
 38. A conjugate according to claim 4, whereinsaid cancer is carcinoma.
 39. A conjugate according to claim 12, whereinthe covalent organic linkage structure B comprises a polypeptide chain.40. A conjugate according to claim 12, wherein the covalent organiclinkage structure B is longer than 3 atoms and shorter than 100 atoms.41. A conjugate according to claim 40, wherein the covalent organiclinkage structure B comprises a polypeptide chain.
 42. A conjugateaccording to claim 41, wherein the polypeptide chain comprises naturallyoccurring hydrophilic alpha amino acids.
 43. A conjugate according toclaim 42, wherein the polypeptide chain comprises amino acids selectedfrom the group consisting of asparagine, glutamine, lysine, arginine,glycine, threonine, serine and histidine.
 44. A conjugate according toclaim 39, wherein the covalent organic linkage structure B has beenrecombinantly produced.
 45. A conjugate according to claim 40, whereinthe covalent organic linkage structure B comprises a polypeptide chaincomprising naturally occurring hydrophilic alpha amino acids selectedfrom the group consisting of asparagine, glutamine, lysine, arginine,glycine, threonine, serine and histidine.
 46. A conjugate according toclaim 1, wherein said target cell is a human cell.
 47. A conjugateaccording to claim 1, wherein said disease is a human disease, and saidtarget cell is a human tumor cell.
 48. A conjugate according to claim 1,wherein said target cell is capable of being induced to express MHCClass II antigens as a consequence of conjugate activation of immunecells.
 49. A conjugate according to claim 1, wherein said antibodycomprises an active antibody fragment.
 50. A conjugate according toclaim 1, wherein the superantigen and antibody are held together througha covalent organic linkage structure B which comprises at least oneamide structure, and comprises a polypeptide chain comprising naturallyoccurring hydrophilic alpha amino acids selected from the groupconsisting of asparagine, glutamine, lysine, arginine, glycine,threonine, serine and histidine.
 51. A method according to claim 16,wherein said antibody comprises an active antibody fragment.
 52. Amethod according to claim 17, wherein said cancer is carcinoma.
 53. Amethod according to claim 25, wherein the covalent organic linkagestructure B comprises a polypeptide chain.
 54. A method according toclaim 25, wherein the covalent organic linkage structure B is longerthan 3 atoms and shorter than 100 atoms.
 55. A method according to claim54, wherein the covalent organic linkage structure B comprises apolypeptide chain.
 56. A method according to claim 55, wherein thepolypeptide chain comprises naturally occurring hydrophilic alpha aminoacids.
 57. A method according to claim 56, wherein the polypeptide chaincomprises amino acids selected from the group consisting of asparagine,glutamine, lysine, arginine, glycine, threonine, serine and histidine.58. A method according to claim 53, wherein the covalent organic linkagestructure B has been recombinantly produced.
 59. A method according toclaim 54, wherein the covalent organic linkage structure B comprises apolypeptide chain comprising naturally occurring hydrophilic alpha aminoacids selected from the group consisting of asparagine, glutamine,lysine, arginine, glycine, threonine, serine and histidine.
 60. A methodaccording to claim 16, wherein said target cell is a human cell.
 61. Amethod according to claim 16, wherein said disease is a human diseaseand said target cell is a human tumor cell.
 62. A method according toclaim 16, wherein said target cell is capable of being induced toexpress MHC Class II antigens as a consequence of conjugate activationof immune cells.
 63. A method according to claim 14, wherein, thesuperantigen and antibody are held together through a covalent organiclinkage structure B which comprises at least one amide structure, andcomprises a polypeptide chain comprising naturally occurring hydrophilicalpha amino acids selected from the group consisting of asparagine,glutamine, lysine, arginine, glycine, threonine, serine and histidine.64. A method according to claim 15, wherein said antibody comprises anactive antibody fragment.
 65. A method according to claim 27, whereinsaid cancer is carcinoma.
 66. A method according to claim 35, whereinthe covalent organic linkage structure B comprises a polypeptide chain.67. A method according to claim 35, wherein the covalent organic linkagestructure B is longer than 3 atoms and shorter than 100 atoms.
 68. Amethod according to claim 67, wherein the covalent organic linkagestructure B comprises a polypeptide chain.
 69. A method according toclaim 68, wherein the polypeptide chain comprises naturally occurringhydrophilic alpha amino acids.
 70. A method according to claim 69,wherein the polypeptide chain comprises amino acids selected from thegroup consisting of asparagine, glutamine, lysine, arginine, glycine,threonine, serine and histidine.
 71. A method according to claim 66,wherein the covalent organic linkage structure B has been recombinantlyproduced.
 72. A method according to claim 67, wherein the covalentorganic linkage structure B comprises a polypeptide chain comprisingnaturally occurring hydrophilic alpha amino acids selected from thegroup consisting of asparagine, glutamine, lysine, arginine, glycine,threonine, serine and histidine.
 73. A method according to claim 15,wherein said target cell is capable of being induced to express MHCClass II antigens